This project aims to test whether target genes of Id1 are required for cancer stem cell viability, self-renewal, and proliferation in triple negative breast cancer. 34 candidate target genes of Id1 were identified through bioinformatic analysis. These target genes are being tested using RNA interference experiments to determine if they are important for the cancer stem cell phenotype and could be potential therapeutic targets. Effective targeting of cancer stem cells is needed to completely eliminate tumors and prevent relapse in patients.

1. Aim
This project aims to test the requirement of target
genes of Id1 for the viability, self-renewal, and
proliferation of cancer stem cells (CSCs). The aim is
thus to test whether these candidates are potential
therapeutic targets in triple negative breast cancer
(TNBC) with the future perspective of developing
targeted treatments.
Id1 expression system Id1 depletion system
RNA-Seq Gene Array
4267 9234
Array data
Id1 KD 4T1
RNA-seq data
Id1+ C3Ttg
Bioinformatic
analyses
siRNA-mediated
knockdown of
target gene
Knockdown of
taget gene affects
self-renewal
Knockdown of taget
gene does not affect
self-renewal
Garvan Institute of Medical Research 384 Victoria Street Darlinghurst NSW 2010 Australia
Cancer Tumor Progression
Acknowledgements: This research is funded by the Cancer Counsel
NSW, Knud Højgaards Fond, Oticon Fonden, and Kræftens
Bekæmpelse.
Christina Konrad, Radhika Nair, Wee Teo, Kate Harvey, Daniel Roden, Ben Elsworth, Alexander Swarbrick
The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Sydney
Deciphering the biology of Cancer Stem Cells in
triple negative breast cancer
Candidate targets of Id1
TocharacterisethenetworkofgenesregulatedbyId1,
bioinformaticanalyseswereperformedongenearray
or RNA-Seq data from two differentTNBC models. 34
high confidence targets of Id1 were identified.
Validation of target genes
The requirement of these candidate genes for the
viability, proliferation, and self-renewal of CSCs is
currently being tested in vitro by knockdown studies
using the tumorsphere and proliferation assays.
Id1 targets that are important for the CSC phenotype
are potential therapeutic targets with the future
prospect of developing targeted treatment for TNBC
patients.
Background
While targeted treatments have resulted in a
significant decrease in mortality rates for Hormone
receptor and Her2 positive subtypes of breast cancer,
no targeted treatment exist for patients with the
aggressive triple negative breast cancer subtype.
Malignant progression in TNBC is known to be
driven by a subpopulation of tumor cells termed
cancer stem cells (CSCs). Effective therapeutic
targeting of CSCs is thus essential for the complete
eradication of a tumor and prevention of relapse in
patients due to outgrowth of chemo resistant CSCs.
Research by our group has shown that the tran-
scriptional repressor, Inhibitor of Differentation (Id1),
is expressed within the CSCs of TNBC tumors and is
important for the CSC phenotype like proliferation,
self-renewal, and metastasis.
Knockdown of Id1/Id3 in a TNBC cell line model results in decreased
cell proliferation.
Knockdown of Id1/Id3 reduces self-renewal in a TNBC cell line model.
Knockdown of Id1/Id3 suppresses spontaneous lung metastasis in
mice.
Self-renewal
Metastasis
Proliferation
siRNA-mediated knockdown
Tumorsphere assay
A key property of CSCs is their ability to self-renew.
This property is tested in the tumorsphere assay in
which cells are exposed to low adherent and non-
differentiating conditions. Only cells with high self-
renewal capacity are able to form tumorspheres
under these conditions and thus have a phenotype
associated with CSCs.
Possible outcomesTumorsphere assayLabeled cells
4T1 tumorsphere assay +/- methylcellulose
Numberofprimary
tumorspheres/1000cells
0
20
40
60
80
Labeled 4T1 cells
+ methylcellulose
Labeled 4T1 cells
- methylcellulose
Parental 4T1 cells
+ methylcellulose
Parental 4T1 cells
- methylcellulose
5000 10,000 5000 10,000 5000 10,000 5000 10,000
4T1 tumorsphere assay +/- methylcellulose
Numberofsecondary
tumorspheres/1000cells
0
20
40
60
80
Labeled 4T1 cells
+ methylcellulose
Labeled 4T1 cells
- methylcellulose
Parental 4T1 cells
+ methylcellulose
Parental 4T1 cells
- methylcellulose
1000 5000 1000 5000 1000 5000 1000 5000
4T1 tumorsphere assay +/- methylcellulose
Numberofsecondary
tumorspheres/1000cells
0
20
40
60
80
Labeled 4T1 cells
+ methylcellulose
Labeled 4T1 cells
- methylcellulose
Parental 4T1 cells
+ methylcellulose
Parental 4T1 cells
- methylcellulose
1000 5000 1000 5000 1000 5000 1000 5000
4T1 tumorsphere assay +/- methylcellulose
Numberofsecondary
tumorspheres/1000cells
0
20
40
60
80
Labeled 4T1 cells
+ methylcellulose
Labeled 4T1 cells
- methylcellulose
Parental 4T1 cells
+ methylcellulose
Parental 4T1 cells
- methylcellulose
1000 5000 1000 5000 1000 5000 1000 5000
Defined shapeIrregular shape
- Methylcellulose + Methylcellulose- Methylcellulose + Methylcellulose
~100% aggregates ~50% aggregates
Seeding number
Methylcellulose
A lower seeding number reduced the number of aggregates. However,
even at the lowest seeding number aggregates were formed.
Addition of 1% methylcellulose to the tumorsphere culture medium
resulted in reduced aggregation and spheres with a more defined
shape.
GFP
RFP
Tumorspheres formed at different seeding
numbers of MDA-MB-231 cells
MDA-MB-231 seeding number
20000
15000
10000
5000
0
20
40
60
80
20000
15000
10000
5000
Numberoftumorspheres
Tumorspheres formed at different seeding
numbers of MDA-MB-231 cells
20000
15000
10000
5000
0
2
4
6
8
20000
15000
10000
5000
MDA-MB-231 seeding number
Numberoftumorspheres/1000cells
Optimization of tumorsphere assay